Recent Advances in the
`Polymerase Chain Reaction
`
`HENRY A. ERLIcH, DAvm GELFAND, JOHN J. SNINSKY
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` on March 27, 2013
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`into the PCR product and mismatches between the primer and the
`original genomic template can be tolerated, new sequence informa-
`tion (specific mutations, restriction sites, regulatory elements) and
`labels can be introduced via the primers into the amplified DNA
`fragment (3-5).
`The ability to amplify as well as modify a specific target DNA
`sequence from a complex template in a simple automated procedure
`has facilitated many tasks in molecular biology research (for exam-
`ple, cloning and sequencing), thus opening up new areas for
`experimental investigation. Here, we review some of the recent
`developments in PCR procedures as well as some recent applica-
`tions.
`
`New Procedures and Reagents
`The initial studies that developed the PCR (1, 2) utilized the
`Klenow fragment of Escherichia coli DNA polymerase I to amplify
`specific targets from human genomic DNA. The inactivation of this
`polymerase at the high temperatures necessary for strand separation
`required the addition of enzyme after the denaturation step of each
`cycle. This rather tedious step was eliminated by the introduction of
`a thermostable DNA polymerase, the Taq DNA polymerase, isolat-
`ed from the thermophilic bacterium Themus aquaticus (6). The use
`of Taq DNA polymerase has transformed the PCR by allowing the
`development of simple automated thermal cycling devices for car-
`rying out the amplification reaction in a single tube containing the
`necessary reagents (7). The availability of a thermostable enzyme has
`not only simplified the procedure for the PCR but has increased the
`specificity and yield of the amplification reaction (7). The incorpo-
`ration of Taq DNA polymerase into the PCR protocol allows the
`primers to be annealed and extended at much higher temperature
`than was possible with Klenow fragment, eliminating much of the
`nonspecific amplification. Moreover, long PCR products could be
`amplified from genomic DNA, probably due to a reduction in the
`secondary structure of the template strands at the elevated temper-
`ature used for primer extension. The upper size limit for amplifica-
`tion with the Klenow fragment was only about 400 bp. Fragments
`as large as 10 kb have been synthesized with Taq DNA polymerase
`and other thermostable enzymes.
`Recent developments in PCR amplification protocols have affect-
`ed critical parameters, including the misincorporation rate, specific-
`ity (target versus nontarget amplification), and maximum length of
`PCR products. Taq DNA polymerase has no 3' to 5' exonuclease
`("proofreading) activity, but has a 5' to 3' exonuclease activity
`during polymerization. The initial estimates of the misincorporation
`rate by Taq DNA polymerase during PCR (about 10-4 nucleotides
`per cycle) were based on measuring the frequency of nucleotide
`substitutions in the sequence analysis of cloned PCR products (7).
`Since then, changes in the PCR conditions such as lower concen-
`ARTICLES
`1643
`
`The polymerase chain reaction (PCR) has dramatically
`altered how molecular studies are conducted as well as
`what questions can be asked. In addition to simplifying
`molecular tasks typically carried out with the use of
`recombinant DNA technology, PCR has allowed a spec-
`trum ofadvances ranging from the identification ofnovel
`genes and pathogens to the quantitation of characterized
`nucleotide sequences. PCR can provide insights into the
`intricacies ofsingle cells as well as the evolution ofspecies.
`Some recent developments in instrumentation, method-
`ology, and applications of the PCR are presented in this
`review.
`
`SINCE ITS INTRODUCMION IN 1985, THE POLYMERASE CHAIN
`reaction (PCR) (1, 2) has transformed the way DNA analysis
`is carried out in both research and clinical laboratories. The
`PCR, which was developed by scientists at Cetus, involves the in
`vitro enzymatic synthesis of millions of copies of a specific DNA
`segment. The reaction is based on the annealing and extension of
`two oligonudeotide primers that flank the target region in duplex
`DNA; after denaturation ofthe DNA, each primer hybridizes to one
`of the two separated strands such that extension from each 3'
`hydroxyl end is directed toward the other. The annealed primers are
`then extended on the template strand with a DNA polymerase.
`These three steps (denaturation, primer binding, and DNA synthe-
`sis) represent a single PCR cycle. Although each step can be carried
`out at a discrete temperature (for example, 940 to 98WC, 37' to 65WC,
`and 720C, respectively), a reaction cycled between the denaturation
`and the primer binding temperatures generally allows sufficient time
`for polymerase activity to amplify short PCR products. If the newly
`synthesized strand extends to or beyond the region complementary
`to the other primer, it can serve as a primer binding site and template
`for a subsequent primer extension reactions. Consequently, repeated
`cycles of denaturation, primer annealing, and primer extension
`result in the exponential accumulation of a discrete fragment whose
`termini are defined by the 5' ends of the primers. This exponential
`amplification results because under appropriate conditions the prim-
`er extension products synthesized in cycle "n" function as templates
`for the other primer in cycle "n + l." The length of the products
`generated during the PCR is equal to the sum of the lengths of the
`two primers plus the distance in the target DNA between the
`primers. PCR can amplify double- (ds) or single-stranded (ss) DNA,
`and with the reverse transcription of RNA into a cDNA copy, RNA
`can also serve as a target. Because the primers become incorporated
`
`The authors are in the Department of Human Genetics, Core Technology, and
`Department of Infectious Diseases, Cetus Corporation, Emeryville, CA 94608.
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`21 JUNE 1991
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`PCR strategies.
`Genetically engineered variants of the thermostable Taq DNA
`polymerase exhibit properties that can be useful for amplifying
`longer PCR products. For example, a mutant Taq DNA polymerase
`lacking the 5' to 3' exonudease (18) permits efficient amplification
`of long fiagments. A truncated Taq DNA polymerase (the Stoffel
`fragment) that lacks the 5' to 3' exonuclease has been generated and
`may reveal properties valuable for particular PCR applications.
`In general, enzymes that have or lack the 5' to 3' and 3' to 5'
`exonuclease activities will perform differently in the PCR. The 5' to
`3' exonuclease of Taq DNA polymerase has been exploited in a
`detection assay based on the cleavage of an oligonudeotide probe
`labeled at the 5' end and blocked at the 3' end. The labeled
`oligonucleotide hybridizes to a target sequence 3' to one primer. In
`the presence of amplified target DNA, the exonuclease activity ofthe
`polymerase leaves a labeled fragment from the 5' end of the
`annealed oligonucleotide during primer elongation (19). What
`results is the release of a sequence-specific signal concomitant with
`amplification.
`Many of the new thermostable polymerases have additional usefiu
`activities. The thermostable DNA polymerase from Themus themo-
`philus (Tth) can reverse-transcribe RNA efficiently in the presence of
`MnCI2 at high temperatures (20). The DNA polymerase activity is
`enhanced by chelating Mn2' and adding MgCI2, allowing the
`cDNA synthesis and PCR amplification to be carried out in a
`single-enzyme, single-tube reaction. In addition, these new thermo-
`stable polymerases may be more resistant to blood components that
`inhibit Taq DNA polymerase.
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`Reaction Specificity
`The initial PCR amplifications with the Klenow fragment were
`not highly specific; although a unique DNA fragment could be
`amplified -200,000-fold from genomic DNA, only about 1% of
`the PCR product was the targeted sequence (3). A specific hybrid-
`ization probe was therefore required to analyze the amplified DNA
`13). Amplification with the Taq DNA polymerase greatly
`(1,
`increased the specificity of the reaction (7), so that for many
`amplifications the PCR products could be detected as a single
`ethidium bromide-stained band on an electrophoretic gel. Condi-
`tions that increase the stringency of primer hybridization, such as
`higher annealing temperatures and lower MgC12 concentrations,
`enhance specific amplification. In addition, the concentration of
`enzyme and primers as well as the annealing time, extension time,
`and number of cycles also effect the specificity of the PCR. The
`concentration of a specific sequence in a sample can also influence
`the relative homogeneity of the PCR products. Thus, a single copy
`nuclear gene present twice in every diploid cell can usually be
`detected as a unique band after gel electrophoresis of the PCR
`products; amplification of a sequence present in only one of 10,000
`cells is likely to yield a more heterogenous gel profile.
`New approaches to improve specificity have been developed.
`These strategies are based on the recognition that the Taq DNA
`polymerase retains considerable enzymatic activity at temperatures
`well below the optimum for DNA synthesis. Thus, in the initial
`heating step of the reaction, primers that anneal nonspecifically to a
`partially single-stranded template region can be extended and stabi-
`lized before the reaction reaches 720C for extension of specifically
`annealed primers. Some of these nonspecifically annealed and ex-
`tended primers may be oriented with their 3' hydroxyl directed
`toward each other, resulting in the exponential amplification of a
`nontarget fragment. If the DNA polymerase is activated only after
`the reaction has reached high (>70WC) temperatures, nontarget
`SCIENCE, VOL. 252
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`trations of dNTP's and MgCl2, higher annealing temperatures, and
`shorter extension times have reduced the misincorporation rate to
`less than 10-5 nucleotides per cycle (8, 9).
`Other DNA polymerases, like the bacteriophage T4 DNA poly-
`merase, appear to have a very low misincorporation rate in PCR
`(10). The heat lability ofthis enzyme, however, limits its utility. The
`thennostable VENT DNA polymerase, an enzyme recently isolated
`from Thermococcus litoralis, has the 3' to 5' exonuclease activity, and
`may therefore have a lower misincorporation rate. Further studies
`will be required to determine if the capacity of this 3' to 5'
`exonuclease activity to degrade single-stranded molecules (like oli-
`gonudeotide primers or PCR product prior to primer annealing)
`will pose problems for PCR amplification. In addition, DNA
`polymerases with 3' to 5' exonuclease activity probably cannot be
`used in sequence-specific priming reactions because this activity
`removes the mismatched base at the 3' end of the primer.
`For most PCR applications, such as direct sequencing (11, 12) or
`oligonucleotide probe typing (13), it is the population of amplifi-
`cation products that is analyzed, and therefore rare misincorporated
`nudeotides are not detected. A specific misincorporation will be
`present in a significant fraction of the PCR products only if the
`insertion of an incorrect base occurs at the first cycle of a reaction
`initiated with very few templates. The determination of individual
`cloned sequences derived from PCR products, however, can reveal
`such rare errors; the analysis of the PCR products by denaturing
`gradient gel electrophoresis has also been used to reveal the presence
`of sequences that contain in vitro mutations (10).
`Another category of rare PCR artifacts revealed initially by
`sequencing cloned PCR amplification products is the hybrid se-
`quences generated by in vitro recombination or template strand
`14, 15). Although
`switching (the so-called "shuffle clones") (7,
`misincorporation is rare and the appearance of hybrid sequences
`even less frequent, it is advisable to sequence multiple clones derived
`from a single amplification reaction or to clone the PCR products
`from several independent amplifications to distinguish the sequence
`of the true genomic template from any potential PCR artifact. In
`amplification reactions from heterozygous individuals, hybrid se-
`quences can result from a primer that has been partially extended on
`one allelic template switching to the other allelic template in a
`subsequent cyde. In the amplification of multigene families, the
`potential for switching to another template is increased. The prob-
`ability of partially extended primers competing successfully with the
`primer is a function of the PCR product concentration. Two recent
`studies on this PCR artifactual recombination may therefore have
`exaggerated its significance for genomic DNA amplification by
`studying systems with very high concentrations of plasmid target
`DNA sequences (14, 15).
`The probability that a primer will not be fully extended (will not
`reach the other primer binding site) is dependent on distance,
`secondary structure, extent of DNA degradation in the template
`(14), enzyme limitation, the time allowed for polymerase extension,
`and the processivity of the polymerase. Ihe use of DNA polymer-
`ases with increased processivity as well as conditions that increase
`processivity (for example, accessory proteins or lower salt concen-
`trations) may aid in achieving full primer extension. More impor-
`tantly, new DNA polymerases may allow the amplification of larger
`PCR products, a development that would be valuable for the
`physical mapping and sequencing aspects of the Human Genome
`Project (16). Richardson and co-workers (17) have shown that a
`12-kD Escherichia coli protein can increase the affinity of the
`bacteriophage T7 DNA polymerase for a primer-template complex,
`conferring a higher degree of processivity on the enzyme. However,
`given the thermal cycling properties of the PCR, only enzymes and
`auxiliary proteins that are heat-resistant are likely to function in new
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`primers to amplify the targeted subset of the initial PCR products.
`This can be accomplished with an outer primer that has a "GC-
`clamp" at the 5' end such that the melting temperature of the initial
`PCR product is high, and with an inner primer that is sufficiently
`short (or AT-rich) so that it cannot bind to the template at the
`annealing temperature used in the initial PCR cycling. If the initial
`PCR conditions involve a thermal profile with elevated denaturation
`and annealing temperatures, then the outer pair of primers but not
`the inner primers will function. After n cycles, the annealing
`temperature is dropped, allowing the inner primer or primers to
`anneal. Then, after n + x cycles the denaturation temperature is
`dropped, preventing the initial PCR product from denaturing and
`serving as a template and a binding site for the outer primers. This
`strategy, termed 'drop-in, drop-out nested priming," has been
`carried out with a single inner nested primer (hemi-nesting) (23),
`resulting in the amplification of a single human immunodeficiency
`virus (HIV) template in the background of genomic DNA from
`-70,000 cells (Fig. 1). The "drop-out" of the outer primer can also
`be accomplished when limiting concentrations of this primer are
`used, as in the strategy of "asymmetric PCR," a protocol for
`generating single strands of DNA (24).
`
`Contamination of PCR Reactions
`Because the PCR can generate trillions of DNA copies from a
`template sequence, contamination of the amplification reaction with
`products of a previous PCR reaction (product carryover), exoge-
`nous DNA, or other cellular material can create problems both in
`research and diagnostic applications. In general, attention to careful
`laboratory procedures-pre-aliquoting reagents, the use of dedicat-
`ed pipettes, positive-displacement pipettes, or tips with barriers
`preventing contamination of the pipette barrel, and the physical
`separation of the reaction preparation from the area of reaction
`product analysis-minimizes the risk of contamination (25). Multi-
`ple negative controls (no template DNA added to the reaction) are
`necessary to monitor and reveal contamination. In genetic typing
`the contamination of a sample reaction can often be detected by a
`genotyping result with more than two alleles.
`Several approaches to mnimi
`the potential for PCR product
`carryover have been developed, all based on interfering with the
`ability of the amplification products to serve as templates. One such
`strategy developed independently by two groups (26, 27) utilizes the
`principles of the restriction-modification and excision repair systems
`of bacteria to pretreat PCR reactions and selectively destroy DNA
`synthesized in a previous PCR. In order to distinguish PCR
`products from sample template DNA, deoxyuridine triphosphate
`(dUTP) is substituted for deoxythymidine triphosphate (dITP) in
`the PCR and is incorporated into the amplification products. The
`presence of this unconventional nucleotide allows the distinction of
`products of previous PCR amplifications from the native DNA of
`the sample. The enzyme uracil N-glycosylase (UNG), present in the
`reaction pre-mix, catalyzes the excision of uracil from any potential
`single- or double-stranded PCR carry-over DNA present in the
`reaction prior to the first PCR cycle. RNA can still serve as a
`template for the PCR because UNG does not excise uracil from
`RNA. The abasic polynucleotides that result from cleavage by UNG
`are susceptible to hydrolysis in alkaline solutions (like PCR buffers)
`and at elevated temperatures. During PCR, the abasic polynucle-
`otides cannot function as templates because of DNA polymerase
`salling or strand scission; the aglycosidic linkage is cleaved at the
`high denaturation temperature of the first PCR cycle. Furthermore,
`the resulting modified 3' termini of the degraded carryover prod-
`ucts are incapable of priming if a DNA polymerase that lacks a 3'
`1645
`ARTICLES
`
`amplification can be minimized (21). This can be accomplished by
`manual addition of an essential reagent (DNA polymerase, magne-
`sium chloride, primers) to the reaction tube at elevated tempera-
`tures, an approach termed "hot start." The addition of E. coli
`ssDNA binding protein has also been reported to increase specific
`amplification (22).
`Hot start not only improves specificity but miniizes the forma-
`tion of the so-called "primer-dimer," a double-stranded PCR prod-
`uct consisting of the two primers and their complementary se-
`quences. This designation may be somewhat misleading, as sequence
`analysis ofsome ofthese products indicates that additional bases are
`inserted between the primers. As a result, a fraction ofthese artifacts
`may be due to spurious nonspecific amplification of similar but
`distinct primer binding regions that are positioned in the immediate
`vicinity. The synthesis of these nonspecific products appears to be a
`function of the primer concentration. The initial formation of these
`products can occur at low (ambient) temperatures; hot start elimi-
`nates this phase of the thermal profile. Single internal nudeotide
`substitutions in one ofthe primers may lead to differences in the size
`and amount of these background products. Once formed, this
`artifactual PCR product is efficiently amplified; it is often detected in
`reactions with rare or no specific template. In fact, the presence of
`this artifact can be used to distinguish reactions that lack template
`from unsuccessful amplifications that result from an inhibitor ofTaq
`DNA polymerase. In rare template reactions, these short amplifica-
`tion products may compete with the target fragment for primers and
`enzyme and prevent efficient target amplification.
`Another approach that can improve PCR specificity is to follow
`the initial amplification reaction with an additional PCR with
`internal, single, or double nested primers. Like the use of oligonu-
`deotide hybridization probes, this approach utilizes sequence infor-
`mation internal to the two outer primers to identify the subset of
`amplification products that corresponds to the target fragment. The
`first use of a nested primer strategy was in the amplification of
`p-globin from human DNA with the Klenow fragment (2). One
`complication of the conventional nesting strategy is that it requires
`opening the reaction tube, eliminating or decreasing the concentra-
`tion of the original outer primers, and adding the inner primers.
`Although this can sometimes be accomplished by simply diluting
`the first reaction into a second one, this procedure is both inconve-
`nient and risky, in that it provides an opportunity for contaminating
`the secondary reaction with amplified product from previous reac-
`tions. This problem can be overcome if the outer and inner primers
`are all present in the initial reaction mix and if the thermal profile is
`programmed to allow the outer primers but not the inner primers to
`amplify initially and then to allow the inner but not the outer
`
`1
`
`6 7 8
`2 3 4 5
`. genome DNA
`AHIV product
`4-pnrmes
`
`Fig. 1. The use of 'drop
`in, drop-out" nested prim-
`ers and "hot start"
`to
`enhance specific amplifica-
`of single-template
`tion
`H1V sequences.
`Single-
`template PCR reactions
`were set up by limiting dilution of cloned HIV gag sequences into human
`genomic DNA (0.5 fig). The expectation ofthe Poisson distribution for the
`concentration used (-0.5 templates per tube) is that four of eight tubes
`would have one template. The use of a thermal profile that dropped the
`annealing temperature by 120C after 29 (n) cycles and decreased the
`denaturation temperature by 10MC after 31 (n + 2) cydes allowed single-
`tube nested primer amplification. Primer extension was initiated by adding
`the primers after the temperature of the reaction had reached 720C. Details
`ofthe reaction and the sequences ofthe three primers were as described (23).
`Lanes 1 and 8 show a single ethidium bromide-stained band generated from
`a single HIV copy present in a highly complex template sample; no other
`PCR products are observed. No products are observed in the other six
`reactions, probably as a result of the absence of specific template.
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`21 JUNE 1991
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`insertion site (7). In general, flanking sequences for priming are
`added by ligation (32) or homopolymer tailing with terminal
`transferase (33). These approaches, termed anchored or single-sided
`PCR (33, 34), have generally been applied to cDNA, bacterial
`genomes, or cloned sequences, all of which are less complex than
`human genomic DNA.
`One strategy for the analysis of unknown sequences that flank a
`region of known sequence (inverse or inside-out PCR) involves the
`digestion of the template with a restriction enzyme that cuts outside
`the known sequence (35-37). The resulting fragment is then circu-
`larized by ligation and amplification is carried out with primers that
`hybridize to the known sequence but whose 3' hydroxyls point away
`from each other. After circularization, the unknown sequences that
`had flanked the known region become joined between the 3' termini
`of the primers and can therefore be amplified and analyzed. A recent
`approach to capturing flanking sequences that is not dependent on
`template circularization is to use a specific target primer and a panel
`of random oligonucleotide primers; specific amplification from the
`target into the flanking region is detected by a primer for the
`sequences on one side of the target primer (38).
`Performing the PCR with generic primers such as those comple-
`mentary to repetitive DNA families represents another approach to
`amplifying unknown sequences. Amplification with primers of
`repetitive DNA sequences dispersed in the human genome has
`proven valuable in the analysis of yeast artificial chromosome (YAC)
`clones and somatic cell hybrid cell lines (39). Alu, Kpn, and other
`interspersed repetitive sequences (IRS) have been used for the
`design of primers and are used either singly, in combination, or with
`vector sequence primers to generate unique patterns of PCR
`products (fingerprints). These patterns can be used to construct
`genome maps from overlapping clones or cell hybrids. Amplification
`carried out with human IRS-PCR primers can be used to amplify,
`from templates of limited complexity, DNA fragments that function
`as hybridization probes or sequencing templates. Fluorescent probes
`generated by Alu-PCR from hybrid cell lines have been used for in
`situ hybridization or "chromosome painting" in both metaphase and
`interphase cells (40). PCR amplification with these repetitive se-
`quence primers can also provide a source of the sequence-tagged
`sites (STS) (41) proposed for the integration ofvarious physical and
`genetic mapping strategies. An approach for sequencing long cloned
`sequences exploits the ability of transposons to integrate at random
`into large DNA fragments that have been cloned into vectors (P1
`bacteriophage, F' bacterial plasmids, and YAC vectors) propagated
`in bacterial and yeast cells. After transposon integration, this elegant
`strategy then uses primers complementary to the transposon se-
`quences to generate amplified DNA fragments from the cloned
`inserts (42, 43). Transposon PCR strategies may help overcome
`some of the limitations of IRS-PCR that arise from nonrandom
`distribution of these repetitive sequence regions. The amplification
`of DNA fragments from genomic DNA has recently been carried
`out with short (10-nucleotide) primers of random or arbitrary
`sequence (44, 45). In some cases, the pattern of amplification
`products, dubbed APPCR (arbitrarily primed PCR) or RAPD
`(random amplified polymorphic DNA), varies between different
`individuals and may, thus, serve as potential genetic markers in
`mapping studies.
`In general, these approaches have uncoupled the ability to amplify
`DNA from the selective extraction of a specific fragment from a
`complex genomic template, which was a hallmark of the initial PCR
`strategies. Experimental strategies for isolating a specific targeted
`sequence can then be applied independently of the PCR amplifica-
`tion. In genomic PCR (32), primer sites are ligated onto a restric-
`tion enzyme digest of genomic DNA and the resulting DNA
`fragments are amplified; specific DNA fragments that bind a partic-
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`exonuclease (such as Taq DNA polymerase) is used. Fortunately,
`UNG is inactivated at temperatures used in PCR, so that the
`amplification products generated during the thermal cycling are
`stable and can accumulate. This elegant strategy for eliminating
`PCR product carry-over still allows amplified DNA to serve as a
`target for probe hybridization and for cloning and sequencing.
`Another approach to PCR product inactivation involves short-
`wavelength ultraviolet irradiation of the reaction mixture prior to
`amplification (28). This strategy, however, requires the addition of
`DNA polymerase and target DNA to the reaction tube after the
`irradiation-inactivation step. An alternative inactivation method
`involves the photochemical modification of the amplified DNA,
`thereby blocking the Taq DNA polymerase from further extension
`after it encounters a modified base in the template strand. Isopsor-
`alen reagents can be present throughout the reaction and phoacti-
`vated after amplification; cydobutane adducts with pyrimidine bases
`in the PCR product are formed, preventing subsequent template
`function (29).
`
`Instruments for the PCR and Product
`Analysis
`In addition to the advances in PCR reagents and protocols, new
`instruments for automated thermal cycling and for analyzing the
`PCR products have been developed. The reaction vessels accommo-
`dated by the first generation of thermal cyclers were standard plastic
`microfuge tubes. Some new thermal cyclers have increased rates of
`heating and cooling and ofheat transfer to modified reaction vessels
`(30). PCR amplification in capillary tubes allows rapid thermal
`cycling and has reduced a complete cycle of denaturation, primer
`annealing, and DNA synthesis to 20 s (31); the speed of the
`temperature changes achieved in this system has allowed the precise
`definition oftemperature optima for each individual step in the PCR
`cycle. For the more convenient but slower commercial thermal
`cycling devices, the use of two-temperature (annealing-extension
`and denaturation) thermal profiles can reduce overall cycling time.
`The new generation of automated thermal cyclers is also faster than
`its predecessors, utilizes thin-walled plastic tubes, and can accom-
`modate more samples. Some of these have a reduced thermal
`gradient across the heating block, resulting in more precise thermal
`profiles. In addition, the requirement for mineral oil to prevent
`evaporation and increase the rate of thermal equilibration has been
`eliminated in some new models. Instruments for the electrophoretic
`analysis of PCR products labeled with fluorescent primers have also
`been introduced.
`
`Research Strategies and Applications
`As the performance of PCR has improved with advances in
`amplification protocols, the strategies for applying it to a variety of
`research problems have increased. Many of these strategies have
`been developed to overcome one of the apparent limitations of
`PCR, namely the requirement for specific sequence information to
`design the amplification primers. Although this requirement still
`allows the characterization of mutations, polymorphisms, and evo-
`lutionary changes in the DNA sequence between the primers, it
`represents a constraint on the use ofPCR to analyze unknown DNA
`sequences. This limitation has been overcome by a variety ofspecific
`strategies. The general approach has been to create primer binding
`sites by adding DNA of a known sequence and was illustrated
`initially by the amplification of unknown cDNA sequences cloned
`into X gtll with primers for the vector sequences that flank the
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`ular protein can be identified in electrophoretic mobility shift assays,
`isolated, and reamplified, thus overcoming the usual losses that
`accompany purification or enrichment procedures.
`Introduction of new sequences via PCR primers. New nucleotide
`sequence information can be introduced into the PCR product by
`addition to or alteration of PCR primers; these include restriction
`enzyme sites as well as specific mutations incorporated for the
`analysis ofstructure-function relations (5). Specific sequences can be
`added to the amplified DNA by PCR that are complementary to
`vector sequences, eliminating the need for restriction enzyme diges-
`tion and ligation (46). Regulatory elements such as promoters and
`translation initiation sites can be added to allow expression of the
`PCR products in in vitro systems (47). The isolation of proteins
`expressed in vitro or in vivo from PCR products has been facilitated
`by the addition of sequences that encode a monoclonal antibody
`epitope (48), thus allowing one-step affinity purification of the
`protein encoded by the amplified DNA.
`Footprinting (analysis of DNA-protein interaction). The in vitro
`analysis of protein-DNA interactions known as footprinting in-
`volves the determination of a pattern ofprotection from chemical or
`enzymatic cleavage conferred by a DNA binding protein onto a
`specific nucleotide sequence. In vivo footprinting identifies the
`guanine bases in a DNA segment that are protected from methyl-
`ation by bound protein. Because only methylated guanosines can be
`cleaved by piperidine, this approach makes possible the determina-
`tion of protein-DNA interaction sites within a cell. However, it is
`difficult to detect these interactions. A modification of the in vivo
`procedure that utilizes PCR amplification with ligated primer sites
`to increase the amount of each cleaved genomic fragment has made
`genomic footprinting much more sensitive and, thus, a highly
`promising method for studying DNA-protein interactions in vivo
`(49). In addition, the insertion ofunconventional bases that modify
`protein recognition may be useful for delineation of specific DNA-
`protein contact points.
`PCR and the Human Genome Project. The PCR promises to be
`important in both the mapping (physical and genetic) and the
`sequencing aspects of the Human Genome Project (16). Many
`physical mapping strategies rely on the use of IRS-PCR to obtain
`fingerprint patterns of PCR-amplified human chromosome frag-
`ments in order to create an overlapping linear array. The sequence
`tagged site (STS) proposal (41) also requires the use of PCR to
`generate the 200- to 500-bp sites unique to a given genomic
`fragment, thus identifying the common element necessary to inte-
`grate various physical and genetic maps. The STS proposal, which
`envisions l